TCO conjugates and methods for delivery of therapeutic agents

ABSTRACT

The present invention provides a method for selective delivery of a therapeutic or diagnostic agent to a targeted organ or tissue by implanting a biocompatible solid support in the patient being linked to a first binding agent, and administering a second binding agent to the patient linked to the therapeutic or diagnostic agent, such that the therapeutic or diagnostic agent accumulates at the targeted organ or tissue.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application is a continuation of PCT Application No.PCT/US2015/020718, filed Mar. 16, 2015, which claims priority to U.S.Provisional Application Nos. 62/083,022, filed Nov. 11, 2014,62/013,994, filed Jun. 18, 2014, and 61/953,294, filed Mar. 14, 2014,each of which is incorporated in its entirety herein for all purposes.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH AND DEVELOPMENT

Not Applicable

BRIEF SUMMARY OF THE INVENTION

In a first embodiment, the present invention provides a solid supportcomposition having a biocompatible solid support, at least one binding,and a linker having from about 1 to about 10 linking atoms, covalentlylinking each binding agent to the biocompatible solid support.

In another embodiment, the present invention provides a bioactivecomposition having a therapeutic or diagnostic agent, a binding agentthat can be trans-cyclooctene or tetrazine, and a linker having fromabout 1 to about 10 linking atoms, covalently linking the binding agentto the therapeutic or diagnostic agent.

In another embodiment, the present invention provides a composition of

In another embodiment, the present invention provides a method forselectively delivering an effective amount of a therapeutic ordiagnostic agent to a first location of a targeted organ or tissue in apatient. The method includes implanting a solid support composition ofthe present invention in the patient at the first location of thetargeted organ or tissue, wherein the solid support composition includesa first binding agent. The method also include administering to thepatient a bioactive composition of the present invention, wherein thebioactive composition includes a second binding agent, and wherein thefirst and second binding agents bind to one another upon contact,thereby selectively delivering the effective amount of the therapeuticor diagnostic agent to the first location of the targeted organ ortissue in the patient.

In another embodiment, the present invention provides a method forselectively delivering an effective amount of a therapeutic ordiagnostic agent to a first location of a targeted organ or tissue in apatient. The method includes implanting in the patient at the firstlocation of the targeted organ or tissue a solid support compositionhaving a biocompatible solid support and a first binding agent linked tothe solid support. The method also includes administering to the patienta bioactive composition having a therapeutic or diagnostic agent, asecond binding agent complementary to the first binding agent, and areleasable linker linking the therapeutic or diagnostic agent and thesecond binding agent, such that the first and second binding agent bindto one another upon contact. The method also includes releasing thetherapeutic or diagnostic agent, thereby delivering the therapeutic ordiagnostic agent to the first location of the targeted organ or tissue.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A and FIG. 1B shows the method of the present invention involvingthe initial injection of a biomaterial covalently attached to TCO. Thisis followed by a systemic injection of a therapeutic agent coupled tothe tetrazine moiety. When the two entities come in close proximity,they react covalently attaching the therapy to the solid carrier andthus localizing the therapeutic agent. A molecular probe was utilizedfor Tz radioprobe 2 that contains radioactive ¹¹¹Indium.

FIG. 2 shows in-vitro studies, pre-weighed gel discs (experimental orcontrol) were mixed with a solution of Tz radioprobe 2 for 10 min or 14hours. During a 10 minute exposure a sample of TCO-Gel 1 bonded 1.78nmoles/g, while the control gel maintained 0.75 nmoles/g of Tzradioprobe 2. In 14 hours the amount of Tz-radioprobe attached tocontrol gel remained constant at 0.86 nmoles/g. The TCO-Gel 1 bound 2.78nmoles/g. Error bars represent the standard deviation of the mean forthree replicates. Star indicates statistical significance by pairedt-test (p-value <0.05).

FIG. 3A and FIG. 3B shows Biodistribution results of Tz radioprobe 2. a,Mice (n=3) bearing subcutaneous TCO-Gel 1 (mean 0.23 g [0.10-0.29 g])and control gel (Mean 0.23 g [0.16-0.25 g]) were administered Tzradioprobe 2 (mean 44 μCi [39.2-48.4 μCi]) via tail vein injection(t=0). b, Mean difference at specific timepoints between TCO-Gel 1 &control gel. Bars indicate % ID/g per organ at specific timepoint (1, 4,24, 48 hr). Error bars represent the standard deviation of the mean forthree replicates. Star indicates statistical significance by pairedt-test (p-value <0.05).

FIG. 4 shows a mouse bearing subcutaneous TCO-Gel 1 (170 mg, green area)and control gel (230 mg, yellow area). 3 hours after implantation Tzradioprobe 2 (1.05 mCi) was delivered via tail vein injection and imagedat 4, 24, 48 hour through SPECT.

FIG. 5 shows synthetic preparation of the solid support compositionTz-Me-gel.

FIG. 6 shows the reaction between the tetrazine solid support andfluorescein linked with TCO through a non-releasable linker. Rrepresents the intact diethyl amine fluorescein moiety, still covalentlyconnected to the solid support.

FIG. 7 shows the reaction between the tetrazine solid support andfluorescein linked with TCO through a releasable linker. As expectedfluorescein diethyl amine (MW: 418.40) and carbon dioxide are releasedfrom the solid support.

FIG. 8 provides a sample of the supernatants after removal of the gelused to extrapolate the amount of unreacted TCO-X-Fluorescein and thekinetics of the reaction between TCO-X-Fluorescein and Tz-Me-gel.Briefly, the method involved: forming and weighing the Tz-gel, adding 1mL of PBS saline containing a predetermined concentration ofTCO-X-Fluorescein (10, 20, 50 nmoles), placing the plate at a well-platemixer (speed 200) for predetermined number of time (10, 50, 100, 180minutes), transferring the supernatant at the specific timepoint toanother plate, and measuring the amount of fluorescence remaining in thesupernatant via an IVIS spectrum machine (Fluorescein excitation at 490nm, emission at 515 nm).

FIG. 9 shows a standard curve fit correlating radiance (fluorescence)and amount of fluorophore based on experimental values with a knownamount of nmoles of TCO-NR-Fluorescein and the radiance detected with anIVIS Spectrum machine (Fluorescein excitation at 490 nm, emission at 515nm).

FIG. 10 shows the decrease of TCO-NR-Fluorescein in the supernatant overtime when the TCO-NR-Fluorescein and Tz-Me-gel are mixed at 37° C.between 0 and 70 minutes.

FIG. 11 shows the theoretical reciprocity of TCO-NR-Fluorescein bound tothe gel and the amount detected in the supernatant that remains unbound.

FIG. 12 shows the calculated amount of bound TCO-NR-Fluoresceinmolecules to Tz-Me-gel based on the data of FIG. 10. Based on the datapresented when a Tz-Me-gel disc was mixed with 50 nmoles ofTCO-NR-Flourescein, 0.66 nmoles of TCO-NR-Fluorescein reacted per 1 mgof Tz-Me-gel after 70 minutes (0.66 nmoles/mg). Even after only 70minutes this is about 200 times higher that the amount achieved withTCO-gel (2.78 nmoles/g) after 14 hours as shown in FIG. 2.

FIG. 13 shows the activity of Tz-Me-gel after incubation at 37° C. for apredetermined amount of time (0-3 days). After the allotted incubation,50 mg gels were challenged with the addition of 50 nmoles ofTCO-NR-Fluorescein for 90 minutes and the amount bound was calculated asin the previous figures.

FIG. 14 shows the expected versus observed amount of unbound moleculesafter addition of TCO-R-fluorescein to Tz-Me-gel after multipletime-points. Given the similarities between the TCO-R-Fluorescein andTCO-NR-Fluorescein, similar reactivity patterns are expected. Theassumption is that the difference in amount of recovered fluorescence inthe supernatant is due to the release of diethylfluorescein asillustrated in FIG. 7.

FIG. 15 shows the total amount of fluorescein released after theaddition of a specific amount of TCO-R-fluorescein (10, 20 & 50 nmoles)to Tz-Me-gel. Furthermore, the specific identity of the compounds in thesupernatant of time point 60 minutes were analyzed with an LC-MS andrevealed the presence of Fluorescein diamine (MW 418.40) as the maincompound, further confirming the presence of the elimination product ofthe Catch & Release reaction of TCO-R-Fluorescein with Tz-Me-gel.

FIG. 16A and FIG. 16B shows the method of the present invention usingthe catch-and-release linker. Briefly, the method involves first thelocalization of a solid carrier modified with click chemistry carrier,in this particular instance tetrazine. Then a cargo modified with acarbamate (green) and a trans-cyclooctene moiety is exposed to thematerial (either in-vitro or in-vivo). Both reagents react in-situ anddepending on the substituents of the tetrazine the cargo is eitherreleased from or immobilized to the solid carrier.

FIG. 17 shows the release kinetics of TCO-R-Rhodamine after exposure toagarose beads modified with different tetrazines. Briefly, Agarose 1 awith heterocyclic substituents at X & Y as shown in FIG. 16, leads to aslow release (red line), presumably due to attenuating by electronwithdrawing groups. On the other hand, an aromatic substituent at X & analkyl substituent at Y lead to fast release of Rhodamine diethyl amine.The values are the result of triplicates.

FIG. 18 shows radiance values for mice at 70 minutes, 5 hours and 3days, without a gel impantation or implanted with Tz-gel, or alginateonly, and then injected with TCO-R-rhodamine or TCO-NR-rhodamine.

FIG. 19A and FIG. 19B shows the MIC of TCO-R-vancomycin for luminescentMSSA (Xen 29).

FIG. 20 shows the NMR for(R,E)-N-(2-aminoethyl)-2-(cyclooct-4-en-1-yloxy)acetamide (5).

FIG. 21 shows the NMR for unmodified alginate gel.

FIG. 22 shows the NMR for TCO-gel 1.

FIG. 23 shows the NMR for Tz-Me-gel.

FIG. 24 shows “Catch & Release” strategy, employing bio-orthogonalTCO-tetrazine chemistry.

FIG. 25 shows the amount of flourescein released after 180 minutes. Thisis calculated as the difference between the expected value of boundfluorophore and the actual experimental value obtained.

FIG. 26 shows peaks of fluorescein diamine ([M+H] 419.1214 & [M+MeOH]453.3417) found on supernatant from the mixture of Tz-Me-gel andTCO-R-Fluorescein after 60 minutes, confirming the expected “catch &release” product by Liquid Chromatography—Mass Spectrometry.

FIG. 27 shows the release kinetics of TCO-R-Fluorescein ([M+H] is571.21) after exposure to agarose beads modified with differenttetrazines. Briefly, Agarose 1 a with heterocyclic substituents at X & Yas shown in FIG. 16, leads to a slow release (MX53, red line),presumably due to attenuating by electron withdrawing groups. On theother hand, an aromatic substituent at X & an alkyl substituent at Ylead to fast release of Rhodamine diethyl amine (MX65, blue line). Theleft hand side shows a dynamic evaluation of the release through massspectroscopy.

FIG. 28 shows setup for the kinetic study of ‘Catch & release’ offluorescein-labeled TCO from tetrazine-modified agarose.

FIG. 29 shows a dynamic evaluation of the release of TCO-R-Rhodamine([M+H] 625.31) through mass spectroscopy. The release product is foundas expected at 473.22.

FIG. 30 shows the increase in fluorescence based on the previous kineticbiodistribution study at each time point comparing negative control (nogel injection) vs the release protocol (Tz-Me-Gel) after injection ofTCO-R-Rhodamine. The bar graph extrapolates the data to a presumed totalamount achieved in 8 hours. The dose reflected by the area under thecurve is almost doubled in the presence of the gel as shown in the“released” column (right) vs the negative control (left).

FIG. 31 shows kinetic multiple dose evaluation of mice. Three mice wereevaluated after each injection of 50 nmoles of TCO-R-Rhodamine throughtail vein injection. The mice were treated as described above theirimages. The “negative control” subject did not receive any gelinjection. The “released” subject received an injection in thesubcutaneous back of Tz-Me-gel. The “gel control” subject received aninjection in the subcutaneous back of alginate gel. The mice wereevaluated with an IVIS Spectrum machine after 5-10 minutes and 110-150minutes after each dose of fluorophore.

FIG. 32 shows the average fluorescence observed at 5-10 minutes vs110-150 minutes after the second, third & fourth injection with 50nmoles of TCO-R-Rhodamine to each of the different experimental groups(negative control=no gel; experimental group=Tz-Me-gel; gelcontrol=unmodified alginate gel). The fluorescence is statisticallyhigher in the experimental group than either in the negative or gelcontrol groups. Compared by paired T-test (n=3).

DETAILED DESCRIPTION I. General

The present invention provides compositions for implanting andadministering to a subject where the compositions use a short linkerthat can also include a releasable component. The releasable linkerallows administration of a greater amount of therapeutic or diagnosticagent to the patient in need thereof.

II. Definitions

“Therapeutic agent” refers to an agent capable of treating and/orameliorating a condition or disease. Representative therapeutic agentsinclude, but are not limited to, paclitaxel, doxorubicin, etoposide,irinotecan, SN-38, cyclosporin A, podophyllotoxin, Carmustine,Amphotericin, Ixabepilone, Patupilone (epothelone class), vancomycin,rapamycin and platinum drugs. The therapeutic agent of the presentinvention also include prodrug forms.

“Diagnostic agent” refers to agents that assist in diagnosing conditionsor diseases. Representative diagnostic agents including imaging agentssuch as paramagnetic agents, optical probes, and radionuclides.Paramagnetic agents imaging agents that are magnetic under an externallyapplied field. Examples of paramagnetic agents include, but are notlimited to, iron particles including nanoparticles. Optical probes arefluorescent compounds that can be detected by excitation at onewavelength of radiation and detection at a second, different, wavelengthof radiation. Optical probes useful in the present invention include,but are not limited to, Cy5.5, Alexa 680, Cy5, DiD(1,1′-dioctadecyl-3,3,3′,3′-tetramethylindodicarbocyanine perchlorate)and DiR (1,1′-dioctadecyl-3,3,3′,3′-tetramethylindotricarbocyanineiodide). Other optical probes include quantum dots. Radionuclides areelements that undergo radioactive decay. Radionuclides useful in thepresent invention include, but are not limited to, ³H, ¹¹C, ¹³N, ¹⁸F,¹⁹F, ⁶⁰Co, ⁶⁴Cu, ⁶⁷Cu, ⁶⁸Ga, ⁸²Rb, ⁹⁰Sr, ⁹⁰Y, ⁹⁹Tc, ^(99m)Tc, ¹¹¹n,¹²³I, ¹²⁴I, ¹²⁵I, ¹²⁹I, ¹³⁷Cs, ¹⁷⁷Lu, ¹⁸⁶Re, ¹⁸⁸Re, ²¹¹At, Rn, Ra, Th,U, Pu and ²⁴¹Am.

“Targeted organ or tissue” refers to an organ or tissue that is beingtargeted for delivery of the therapeutic or diagnostic agent.Representative organs and tissues for targeting include those that canbe targeted by chemical or biological targeting agents, as well as thoseorgans and tissues that cannot be targeted by chemical or biologicaltargeting agents. Representative organs or tissues include bone.

“Selectively delivering” refers to delivering a therapeutic ordiagnostic agent to a portion of an organ or tissue in need oftreatment, without targeting other portions of the organ or tissue notin need of treatment.

“Implanting” refers to surgical implantation into the patient's body.

“Biocompatible solid support” refers a solid support material capable ofimplantation into the patient's body and supporting one of the bindingagents, as well as the therapeutic or diagnostic agent after the bindingagents conjugate. The solid support is compatible with the patient'sbody. Representative biocompatible solid supports include, but are notlimited to, hydrogels such as polysaccharide hydrogels, alginate,cellulose, chitosan, hyaluronic acid, chondroitin sulfate, heparin, andothers.

“Contacting” or “contact” refers to the process of bringing into contactat least two distinct species such that they can react. It should beappreciated, however, the resulting reaction product can be produceddirectly from a reaction between the added reagents or from anintermediate from one or more of the added reagents which can beproduced in the reaction mixture.

“Linker”, “linked” or “linking” refers to a chemical moiety that linksthe compound of the present invention to a biological material thattargets a specific type of cell, such as a cancer cell, other type ofdiseased cell, or a normal cell type. The linking can be via covalent orionic bond formation. The linking can be direct linkage between to thetwo moieties being linked, or indirectly, such as via a linker. Linkersuseful in the present invention can be up to 30 carbon atoms in length.Preferably, the linkers are 5-15 carbon atoms in length. The types ofbonds used to link the linker to the compound and biological molecule ofthe present invention include, but are not limited to, amides, amines,esters, carbamates, ureas, thioethers, thiocarbamates, thiocarbonate andthioureas. One of skill in the art will appreciate that other types ofbonds are useful in the present invention.

“Binding agent” refers to any group capable of forming a covalent bondto another binding agent in a biological environment. This is oftenreferred to as bioconjugation or bioorthogonal chemistry. Representativebinding agents include, but are not limited to, an amine and anactivated ester, an amine and an isocyanate, an amine and anisothiocyanate, thiols for formation of disulfides, an aldehyde andamine for enamine formation, an azide for formation of an amide via aStaudinger ligation, an azide and alkyne for formation of a triazole viaClick-chemistry, trans-cyclooctene (TCO) and tetrazine, and others. Thebinding agents useful in the present invention have a high reactivitywith the corresponding binding agent so that the reaction is rapid.

“Treat”, “treating” and “treatment” refers to any indicia of success inthe treatment or amelioration of an injury, pathology, condition, orsymptom (e.g., pain), including any objective or subjective parametersuch as abatement; remission; diminishing of symptoms or making thesymptom, injury, pathology or condition more tolerable to the patient;decreasing the frequency or duration of the symptom or condition; or, insome situations, preventing the onset of the symptom or condition. Thetreatment or amelioration of symptoms can be based on any objective orsubjective parameter; including, e.g., the result of a physicalexamination.

“Administering” refers to oral administration, administration as asuppository, topical contact, parenteral, intravenous, intraperitoneal,intramuscular, intralesional, intranasal or subcutaneous administration,intrathecal administration, or the implantation of a slow-release devicee.g., a mini-osmotic pump, to the subject.

“Patient” refers to animals in need of treatment, such as mammals,including, but not limited to, primates (e.g., humans), cows, sheep,goats, horses, dogs, cats, rabbits, rats, mice and the like. In certainembodiments, the patient is a human.

“Therapeutically effective amount or dose” or “therapeuticallysufficient amount or dose” or “effective or sufficient amount or dose”refer to a dose that produces therapeutic effects for which it isadministered. The exact dose will depend on the purpose of thetreatment, and will be ascertainable by one skilled in the art usingknown techniques (see, e.g., Lieberman, Pharmaceutical Dosage Forms(vols. 1-3, 1992); Lloyd, The Art, Science and Technology ofPharmaceutical Compounding (1999); Pickar, Dosage Calculations (1999);and Remington: The Science and Practice of Pharmacy, 20th Edition, 2003,Gennaro, Ed., Lippincott, Williams & Wilkins). In sensitized cells, thetherapeutically effective dose can often be lower than the conventionaltherapeutically effective dose for non-sensitized cells.

III. Compositions

The present invention provides compositions having a short linker or areleasable linker for selectively delivering a therapeutic or diagnosticagent to a patient. In some embodiments, the present invention providesa solid support composition having a biocompatible solid support, atleast one binding agent, and a linker having from about 1 to about 10linking atoms, covalently linking each binding agent to thebiocompatible solid support.

Any suitable biocompatible solid support can be used in the method ofthe present invention. For example, the biocompatible solid support canbe a hydrogel, a cross-linked polymer matrix, a metal, a ceramic, aplastic, among others. Hydrogels useful in the present inventioninclude, but are not limited to, polysaccharide hydrogels, alginate,cellulose, hyaluronic acid, chitosan, chitosin, chitin, hyaluronic acid,chondroitin sulfate, heparin, and others. Other sugar-based biomaterialsare known in the art, such as those described in Polymer AdvancedTechnology 2014, 25, 448-460. Polymers useful as the biocompatiblesupport can include, but are not limited to, polyphosphazenes,polyanhydrides, polyacetals, poly(ortho esters), polyphosphoesters,polycaprolactones, polyurethanes, polylactides, polycarbonates,polyamides, and polyethers, and blends/composites/co-polymers thereof.Representative polyethers include, but are not limited to, Poly(ethyleneglycol) (PEG), poly(propylene glycol) (PPG), triblock Pluronic([PEG]n-[PPG]m-[PEG]n), PEG diacrylate (PEGDA) and PEG dimethacrylate(PEGDMass.). The biocompatible solid support can also include proteinsand other poly(amino acids) such as collagen, gelatin, elastin andelastin-like polypeptides, albumin, fibrin, poly(gamma-glutamic acid),poly(L-lysine), poly(L-glutamic acid), and poly(aspartic acid).

In some embodiments, the solid support can be a hydrogel. In someembodiments, the solid support can be alginate. In some embodiments, thesolid support can be chitin. In some embodiments, the solid support canbe hyaluronic acid. In some embodiments, the solid support can bechitosin. In some embodiments, the solid support can be agarose.

Any suitable linker can be used in the present invention to link thebinding agent to the biocompatible solid support or the therapeutic ordiagnostic agent. Representative linkers can have about 1 to about 100linking atoms, and can include ethylene-oxy moieties, amines, esters,amides, ketone, urea, carbamate and carbonate functional groups. Otherlinkers useful in the methods of the present invention can have fromabout 1 to about 50 linking atoms, or from about 1 to about 10 linkingatoms, or from about 5 to about 10 linking atoms. Representative linkersinclude, but are not limited to, those shown below:

Other linkers suitable in the present invention include:

Any suitable binding agent can be used in the method of the presentinvention. Representative binding agents can be found in “BioconjugateTechniques” Greg T. Hermanson, 1996 and ACS Chemical Biology 2014, 9,592-605. For example, binding agents useful in the method of the presentinvention include, but are not limited to, cyclooctene, tetrazine,azide, alkyne, amine, activated ester, isocyanate, isothiocyanate,thiol, aldehyde, amide, and others. In some embodiments, the bindingagent can be cyclooctene, tetrazine, azide or alkyne. In someembodiments, the binding agent can be trans-cyclooctene or1,2,4,5-tetrazine. In some embodiments, the binding agent can betrans-cyclooctene. In some embodiments, the binding agent can be4-methyl-1,2,4,5-tetrazine.

In some embodiments, the binding agent and the linker together have thestructure of:

In some embodiments, the composition can have the structure of:

The present invention also provides compositions of a therapeutic agentor diagnostic agent linked to a binding agent via a linker. In someembodiments, the present invention provides a bioactive compositionhaving a therapeutic or diagnostic agent, a binding agent that can betrans-cyclooctene or tetrazine, and a linker having from about 1 toabout 10 linking atoms, covalently linking the binding agent to thetherapeutic or diagnostic agent.

Any therapeutic or diagnostic agent can be used in the method of thepresent invention. Representative therapeutic agents include, but arenot limited to, antibiotics such as vancomycin, paclitaxel, doxorubicin,etoposide, irinotecan, SN-38, cyclosporin A, podophyllotoxin,Carmustine, Amphotericin, Ixabepilone, Patupilone (epothelone class),rapamycin and platinum drugs. Other therapeutic agents includedoxycyclin and other MMP inhibitors. Still other therapeutic agentsinclude daptomycin, L-dopa, oseltamivir, cefalexin, 5-aminolevulinicacid, cysteine, nystatin, amphotericin B, flucytosine, emtricitabine,trimethoprim, sulfamethoxazole, acyclovir, celecoxib, nimodipine,doxycycline, ceftriazone, among others. In some embodiments, thetherapeutic agent can be vancomycin. In other embodiments, thetherapeutic agent can be daptomycin. In yet other embodiment thetherapeutic agent can be doxorubicin. In another embodiment, thetherapeutic agent can be cyclic-adenosine monophosphatidyl (c-AMP).

Representative diagnostic agents including imaging agents such asparamagnetic agents, optical probes, and radionuclides. Paramagneticagents imaging agents that are magnetic under an externally appliedfield. Examples of paramagnetic agents include, but are not limited to,iron particles including nanoparticles. Optical probes are fluorescentcompounds that can be detected by excitation at one wavelength ofradiation and detection at a second, different, wavelength of radiation.Optical probes useful in the present invention include, but are notlimited to, Cy5.5, Alexa 680, Cy5, DiD(1,1′-dioctadecyl-3,3,3′,3′-tetramethylindodicarbocyanine perchlorate)and DiR (1,1′-dioctadecyl-3,3,3′,3′-tetramethylindotricarbocyanineiodide). Other optical probes include quantum dots. Radionuclides areelements that undergo radioactive decay. Radionuclides useful in thepresent invention include, but are not limited to, ³H, ¹¹C, ¹³N, ¹⁸F,¹⁹F, ⁶⁰Co, ⁶⁴Cu, ⁶⁷Cu, ⁶⁸Ga, ⁸²Rb, ⁹⁰Sr, ⁹⁰Y, ⁹⁹Tc, ^(99m)Tc, ¹¹¹In,¹²³I, ¹²⁴I, ¹²⁵I, ¹²⁹I, ¹³¹I, ¹³⁷Cs, ¹⁷⁷Lu, ¹⁸⁶Re, ¹⁸⁸Re, ²¹¹At, Rn, Ra,Th, U, Pu and ²⁴¹Am. The diagnostic agents can also include chelatorssuch as 1,4,8,11-tetraazacyclododecane-1,4,8,11-tetraacetic acid (TETA),4,11-bis(carboxymethyl)-1,4,8,11-tetraazabicyclo[6.6.2] hexadecane(CB-TE2A), diethylenetriaminepentaacetice acid (DTPA) and1,4,7,10-tetra-azacyclodecanetetraacetic acid (DOTA). Other chelatorsare useful in the method of the present invention.

Any suitable linker can be used in the present invention to link thebinding agent to the biocompatible solid support or the therapeutic ordiagnostic agent. Representative linkers can have about 5 to about 100linking atoms, and can include ethylene-oxy moieties, amines, esters,amides, ketone, urea, carbamate and carbonate functional groups. Otherlinkers useful in the methods of the present invention can have fromabout 5 to about 50 linking atoms, or from about 1 to about 10 linkingatoms, or from about 5 to about 10 linking atoms. Representative linkersinclude, but are not limited to, those shown below:

Any suitable binding agent can be used in the method of the presentinvention. Representative binding agents can be found in “BioconjugateTechniques” Greg T. Hermanson, 1996 and ACS Chemical Biology 2014, 9,592-605. For example, binding agents useful in the method of the presentinvention include, but are not limited to, cyclooctene, tetrazine,azide, alkyne, amine, activated ester, isocyanate, isothiocyanate,thiol, aldehyde, amide, and others. In some embodiments, the bindingagent can be cyclooctene, tetrazine, azide or alkyne. In someembodiments, the binding agent can be trans-cyclooctene or1,2,4,5-tetrazine. In some embodiments, the binding agent can betrans-cyclooctene. In some embodiments, the binding agent can be4-methyl-1,2,4,5-tetrazine.

In some embodiments, the binding agent and the linker together have thestructure of:

In some embodiments, the present invention provides a composition of

IV. Method for Selectively Delivering Therapeutic or Diagnostic Agent

The present invention provides a method for selectively delivering atherapeutic or diagnostic agent by implanting the solid supportcomposition of the present invention in a patient in need thereof,followed by administering to the patient the bioactive composition ofthe present invention. In some embodiments, the present inventionprovides a method for selectively delivering an effective amount of atherapeutic or diagnostic agent to a first location of a targeted organor tissue in a patient. The mehtod includes implanting a solid supportcomposition of the present invention in the patient at the firstlocation of the targeted organ or tissue, wherein the solid supportcomposition includes a first binding agent. The method also includeadministering to the patient a bioactive composition of the presentinvention, wherein the bioactive composition includes a second bindingagent, and wherein the first and second binding agents bind to oneanother upon contact, thereby selectively delivering the effectiveamount of the therapeutic or diagnostic agent to the first location ofthe targeted organ or tissue in the patient.

Any suitable organ or tissue can be targeted using the method of thepresent invention. Representative organs or tissues include, but are notlimited to, bone, cartilage, ligaments, tendons, intestines, muscles,nervous system including brain, spinal cord, heart, and nerves, andothers. For example, when the organ is the heart, the method of thepresent invention can be used for cardiac repair. In some embodiments,the first location of the targeted organ or tissue cannot be selectivelytargeted by chemical or biological targeting agents over other locationsof the targeted organ or tissue in the patient. In some embodiments, thetargeted organ or tissue can be bone.

Binding agents suitable in the method of the present invention aredescribed in more detail above for the solid support composition and thebioactive composition. In some embodiments, the first binding agent canbe trans-cyclooctene and the second binding agent can be tetrazine. Insome embodiments, the second binding agent can be 1,2,4,5-tetrazine. Insome embodiments, the second binding agent can be4-methyl-1,2,4,5-tetrazine.

In some embodiments, the implantable composition can have the structure:

In some embodiments, the implantable composition can be:

and the bioactive composition can be:

wherein R can be the therapeutic agent or the diagnostic agent.

The biocompatible solid support can be implanted by any means known toone of skill in the art.

The therapeutic or diagnostic agent can be administered in any suitableamount sufficient to treat the disease or condition the patient issuffering from. The dose, frequency and timing of such administeringwill depend in large part on the selected therapeutic agent, the natureof the condition being treated, the condition of the subject includingage, weight and presence of other conditions or disorders, theformulation being administered and the discretion of the attendingphysician. Generally, the therapeutic or diagnostic agents areadministered in dosages ranging from about 2 mg up to about 2,000 mg perday, although variations will necessarily occur depending, as notedabove, on the disease target, the patient, and the route ofadministration. The therapeutic or diagnostic agents of the presentinvention can be administered as frequently as necessary, includinghourly, daily, weekly or monthly. The therapeutic or diagnostic agentsutilized in the pharmaceutical method of the invention are administeredat the initial dosage of about 0.0001 mg/kg to about 1000 mg/kg daily. Adaily dose range of about 0.01 mg/kg to about 500 mg/kg, or about 0.1mg/kg to about 200 mg/kg, or about 1 mg/kg to about 100 mg/kg, or about10 mg/kg to about 50 mg/kg, can be used. The dosages, however, can bevaried depending upon the requirements of the patient, the severity ofthe condition being treated, and the compound being employed. Forexample, dosages can be empirically determined considering the type andstage of disease diagnosed in a particular patient. The doseadministered to a patient, in the context of the present inventionshould be sufficient to effect a beneficial therapeutic response in thepatient over time, but is typically lower than the dose required totreat the patient without having implanted the biocompatible solidsupport that concentrates the therapeutic or diagnostic agent at theorgan or tissue requiring treatment. The size of the dose also will bedetermined by the existence, nature, and extent of any adverseside-effects that accompany the administration of a particular compoundin a particular patient. Determination of the proper dosage for aparticular situation is within the skill of the practitioner. Generally,treatment is initiated with smaller dosages which are less than theoptimum dose of the compound. Thereafter, the dosage is increased bysmall increments until the optimum effect under circumstances isreached. For convenience, the total daily dosage can be divided andadministered in portions during the day, if desired. Doses can be givendaily, or on alternate days, as determined by the treating physician.Doses can also be given on a regular or continuous basis over longerperiods of time (weeks, months or years).

In some embodiments, the concentration of the therapeutic or diagnosticagent at the first location of the targeted organ or tissue is greaterthan the concentration elsewhere in the patient. In some embodiments,the therapeutic agent is vancomycin.

In another embodiment, the present invention provides a method forselectively delivering an effective amount of a therapeutic ordiagnostic agent to a first location of a targeted organ or tissue in apatient. The method includes implanting in the patient at the firstlocation of the targeted organ or tissue a solid support compositionhaving a biocompatible solid support and a first binding agent linked tothe solid support. The method also includes administering to the patienta bioactive composition having a therapeutic or diagnostic agent, asecond binding agent complementary to the first binding agent, and areleasable linker linking the therapeutic or diagnostic agent and thesecond binding agent, such that the first and second binding agent bindto one another upon contact. The method also includes releasing thetherapeutic or diagnostic agent, thereby delivering the therapeutic ordiagnostic agent to the first location of the targeted organ or tissue.

V. Formulation

The compositions of the present invention can be prepared in a widevariety of oral, parenteral and topical dosage forms. Oral preparationsinclude tablets, pills, powder, dragees, capsules, liquids, lozenges,cachets, gels, syrups, slurries, suspensions, etc., suitable foringestion by the patient. The compositions of the present invention canalso be administered by injection, that is, intravenously,intramuscularly, intracutaneously, subcutaneously, intraduodenally, orintraperitoneally. Also, the compositions described herein can beadministered by inhalation, for example, intranasally. Additionally, thecompositions of the present invention can be administered transdermally.The compositions of this invention can also be administered byintraocular, intravaginal, and intrarectal routes includingsuppositories, insufflation, powders and aerosol formulations (forexamples of steroid inhalants, see Rohatagi, J, Clin. Pharmacol.35:1187-1193, 1995; Tjwa, Ann. Allergy Asthma Immunol. 75:107-111,1995). Accordingly, the present invention also provides pharmaceuticalcompositions including a pharmaceutically acceptable carrier orexcipient and the compounds of the present invention.

For preparing pharmaceutical compositions from the compounds of thepresent invention, pharmaceutically acceptable carriers can be eithersolid or liquid. Solid form preparations include powders, tablets,pills, capsules, cachets, suppositories, and dispersible granules. Asolid carrier can be one or more substances, which may also act asdiluents, flavoring agents, binders, preservatives, tabletdisintegrating agents, or an encapsulating material. Details ontechniques for formulation and administration are well described in thescientific and patent literature, see, e.g., the latest edition ofRemington's Pharmaceutical Sciences, Maack Publishing Co, Easton Pa.(“Remington's”).

In powders, the carrier is a finely divided solid, which is in a mixturewith the finely divided active component. In tablets, the activecomponent is mixed with the carrier having the necessary bindingproperties in suitable proportions and compacted in the shape and sizedesired. The powders and tablets preferably contain from 5% or 10% to70% of the compounds of the present invention.

Suitable solid excipients include, but are not limited to, magnesiumcarbonate; magnesium stearate; talc; pectin; dextrin; starch;tragacanth; a low melting wax; cocoa butter; carbohydrates; sugarsincluding, but not limited to, lactose, sucrose, mannitol, or sorbitol,starch from corn, wheat, rice, potato, or other plants; cellulose suchas methyl cellulose, hydroxypropylmethyl-cellulose, or sodiumcarboxymethylcellulose; and gums including arabic and tragacanth; aswell as proteins including, but not limited to, gelatin and collagen. Ifdesired, disintegrating or solubilizing agents may be added, such as thecross-linked polyvinyl pyrrolidone, agar, alginic acid, or a saltthereof, such as sodium alginate.

Dragee cores are provided with suitable coatings such as concentratedsugar solutions, which may also contain gum arabic, talc,polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and/or titaniumdioxide, lacquer solutions, and suitable organic solvents or solventmixtures. Dyestuffs or pigments may be added to the tablets or drageecoatings for product identification or to characterize the quantity ofactive compound (i.e., dosage). Pharmaceutical preparations of theinvention can also be used orally using, for example, push-fit capsulesmade of gelatin, as well as soft, sealed capsules made of gelatin and acoating such as glycerol or sorbitol. Push-fit capsules can contain thecompounds of the present invention mixed with a filler or binders suchas lactose or starches, lubricants such as talc or magnesium stearate,and, optionally, stabilizers. In soft capsules, the compounds of thepresent invention may be dissolved or suspended in suitable liquids,such as fatty oils, liquid paraffin, or liquid polyethylene glycol withor without stabilizers.

For preparing suppositories, a low melting wax, such as a mixture offatty acid glycerides or cocoa butter, is first melted and the compoundsof the present invention are dispersed homogeneously therein, as bystirring. The molten homogeneous mixture is then poured into convenientsized molds, allowed to cool, and thereby to solidify.

Liquid form preparations include solutions, suspensions, and emulsions,for example, water or water/propylene glycol solutions. For parenteralinjection, liquid preparations can be formulated in solution in aqueouspolyethylene glycol solution.

Aqueous solutions suitable for oral use can be prepared by dissolvingthe compounds of the present invention in water and adding suitablecolorants, flavors, stabilizers, and thickening agents as desired.Aqueous suspensions suitable for oral use can be made by dispersing thefinely divided active component in water with viscous material, such asnatural or synthetic gums, resins, methylcellulose, sodiumcarboxymethylcellulose, hydroxypropylmethylcellulose, sodium alginate,polyvinylpyrrolidone, gum tragacanth and gum acacia, and dispersing orwetting agents such as a naturally occurring phosphatide (e.g.,lecithin), a condensation product of an alkylene oxide with a fatty acid(e.g., polyoxyethylene stearate), a condensation product of ethyleneoxide with a long chain aliphatic alcohol (e.g., heptadecaethyleneoxycetanol), a condensation product of ethylene oxide with a partialester derived from a fatty acid and a hexitol (e.g., polyoxyethylenesorbitol mono-oleate), or a condensation product of ethylene oxide witha partial ester derived from fatty acid and a hexitol anhydride (e.g.,polyoxyethylene sorbitan mono-oleate). The aqueous suspension can alsocontain one or more preservatives such as ethyl or n-propylp-hydroxybenzoate, one or more coloring agents, one or more flavoringagents and one or more sweetening agents, such as sucrose, aspartame orsaccharin. Formulations can be adjusted for osmolarity.

Also included are solid form preparations, which are intended to beconverted, shortly before use, to liquid form preparations for oraladministration. Such liquid forms include solutions, suspensions, andemulsions. These preparations may contain, in addition to the activecomponent, colorants, flavors, stabilizers, buffers, artificial andnatural sweeteners, dispersants, thickeners, solubilizing agents, andthe like.

In another embodiment, the compositions of the present invention can beformulated for parenteral administration, such as intravenous (IV)administration or administration into a body cavity or lumen of anorgan. The formulations for administration will commonly comprise asolution of the compositions of the present invention dissolved in apharmaceutically acceptable carrier. Among the acceptable vehicles andsolvents that can be employed are water and Ringer's solution, anisotonic sodium chloride. In addition, sterile fixed oils canconventionally be employed as a solvent or suspending medium. For thispurpose any bland fixed oil can be employed including synthetic mono- ordiglycerides. In addition, fatty acids such as oleic acid can likewisebe used in the preparation of injectables. These solutions are sterileand generally free of undesirable matter. These formulations may besterilized by conventional, well known sterilization techniques. Theformulations may contain pharmaceutically acceptable auxiliarysubstances as required to approximate physiological conditions such aspH adjusting and buffering agents, toxicity adjusting agents, e.g.,sodium acetate, sodium chloride, potassium chloride, calcium chloride,sodium lactate and the like. The concentration of the compositions ofthe present invention in these formulations can vary widely, and will beselected primarily based on fluid volumes, viscosities, body weight, andthe like, in accordance with the particular mode of administrationselected and the patient's needs. For IV administration, the formulationcan be a sterile injectable preparation, such as a sterile injectableaqueous or oleaginous suspension. This suspension can be formulatedaccording to the known art using those suitable dispersing or wettingagents and suspending agents. The sterile injectable preparation canalso be a sterile injectable solution or suspension in a nontoxicparenterally-acceptable diluent or solvent, such as a solution of1,3-butanediol.

In another embodiment, the formulations of the compositions of thepresent invention can be delivered by the use of liposomes which fusewith the cellular membrane or are endocytosed, i.e., by employingligands attached to the liposome, or attached directly to theoligonucleotide, that bind to surface membrane protein receptors of thecell resulting in endocytosis. By using liposomes, particularly wherethe liposome surface carries ligands specific for target cells, or areotherwise preferentially directed to a specific organ, one can focus thedelivery of the compositions of the present invention into the targetcells in vivo. (See, e.g., Al-Muhammed, J. Microencapsul. 13:293-306,1996; Chonn, Curr. Opin. Biotechnol. 6:698-708, 1995; Ostro, Am. J.Hosp. Pharm. 46:1576-1587, 1989).

VI. Administration

The compositions of the present invention can be delivered by anysuitable means, including oral, parenteral and topical methods.Transdermal administration methods, by a topical route, can beformulated as applicator sticks, solutions, suspensions, emulsions,gels, creams, ointments, pastes, jellies, paints, powders, and aerosols.

The pharmaceutical preparation is preferably in unit dosage form. Insuch form the preparation is subdivided into unit doses containingappropriate quantities of the compounds of the present invention. Theunit dosage form can be a packaged preparation, the package containingdiscrete quantities of preparation, such as packeted tablets, capsules,and powders in vials or ampoules. Also, the unit dosage form can be acapsule, tablet, cachet, or lozenge itself, or it can be the appropriatenumber of any of these in packaged form.

The compound of the present invention can be present in any suitableamount, and can depend on various factors including, but not limited to,weight and age of the subject, state of the disease, etc. Suitabledosage ranges for the compound of the present invention include fromabout 0.1 mg to about 10,000 mg, or about 1 mg to about 1000 mg, orabout 10 mg to about 750 mg, or about 25 mg to about 500 mg, or about 50mg to about 250 mg. Suitable dosages for the compound of the presentinvention include about 1 mg, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90,100, 200, 300, 400, 500, 600, 700, 800, 900 or 1000 mg.

The compounds of the present invention can be administered at anysuitable frequency, interval and duration. For example, the compound ofthe present invention can be administered once an hour, or two, three ormore times an hour, once a day, or two, three, or more times per day, oronce every 2, 3, 4, 5, 6, or 7 days, so as to provide the preferreddosage level. When the compound of the present invention is administeredmore than once a day, representative intervals include 5, 10, 15, 20,30, 45 and 60 minutes, as well as 1, 2, 4, 6, 8, 10, 12, 16, 20, and 24hours. The compound of the present invention can be administered once,twice, or three or more times, for an hour, for 1 to 6 hours, for 1 to12 hours, for 1 to 24 hours, for 6 to 12 hours, for 12 to 24 hours, fora single day, for 1 to 7 days, for a single week, for 1 to 4 weeks, fora month, for 1 to 12 months, for a year or more, or even indefinitely.

The compounds of the present invention can be co-administered withanother active agent. Co-administration includes administering thecompound of the present invention and active agent within 0.5, 1, 2, 4,6, 8, 10, 12, 16, 20, or 24 hours of each other. Co-administration alsoincludes administering the compound of the present invention and activeagent simultaneously, approximately simultaneously (e.g., within about1, 5, 10, 15, 20, or 30 minutes of each other), or sequentially in anyorder. Moreover, the compound of the present invention and the activeagent can each be administered once a day, or two, three, or more timesper day so as to provide the preferred dosage level per day.

In some embodiments, co-administration can be accomplished byco-formulation, i.e., preparing a single pharmaceutical compositionincluding both the compound of the present invention and the activeagent. In other embodiments, the compound of the present invention andthe active agent can be formulated separately.

The compound of the present invention and the active agent can bepresent in the compositions of the present invention in any suitableweight ratio, such as from about 1:100 to about 100:1 (w/w), or about1:50 to about 50:1, or about 1:25 to about 25:1, or about 1:10 to about10:1, or about 1:5 to about 5:1 (w/w). The compound of the presentinvention and the other active agent can be present in any suitableweight ratio, such as about 1:100 (w/w), 1:50, 1:25, 1:10, 1:5, 1:4,1:3, 1:2, 1:1, 2:1, 3:1, 4:1, 5:1, 10:1, 25:1, 50:1 or 100:1 (w/w).Other dosages and dosage ratios of the compound of the present inventionand the active agent are suitable in the compositions and methods of thepresent invention.

VII. Examples

Materials. All reagents and NMR solvents were purchased fromSigma-Aldrich (St. Louis, Miss.), unless otherwise noted. Compound 2 wasobtained from Iris Biotech (Marktredwitz, Germany), while compound 8 waspurchased from Polypure (Oslo, Norway). DOTA-NHS ester was obtained fromMacrocyclics (Dallas, Tex.). Silica gel was purchased from Silicycle(Quebec, Canada), while preparative TLC plates (20×20 cm; 1000 μm inthickness) were purchased from Analtech (Newark, Del.). Ultrapurealginates were purchased from ProNova Biomedical (Norway). [¹¹¹In]Indium chloride solutions was purchased from PerkinElmer (Waltham, US).[⁶⁴Cu] Copper chloride in dilute HCl was purchased from WashingtonUniversity (St. Louis, Miss.) or was produced in-house by the64Ni(p,n)64Cu nuclear reaction using an 11 MeV Siemens RDS 111 cyclotronand purified by anion exchange chromatography (Biorad AG 1-X8).Dulbecco's Phosphate Buffered Saline (DPBS) was purchased fromInvitrogen Corporation (Carlsbad, Calif.).

Methods. NMR experiments were carried out in CDCL₃ or [D₆]DMSO, using aVarian 400 MHz VNMRS machine. High resolution ESI mass spectrometry datawas obtained using Agilent Ion Trap LC/MSD SL at Boston UniversityChemical Instrumentation Center measured either in the positive ornegative. During the organic synthesis phase, an Agilent 1100 Seriessystem equipped with a Waters XBridge C18 Column (19×250 mm) applying agradient of water and MeCN containing 0.1% TFA was used for HPLCpurification.

During radiochemistry and for in-vivo analyses and purifcations,reversed-phase HPLC was performed using a Beckman-Colter System Gold 128(Brea, Calif.) chromatography systems equipped with Jupiter Proteo C-12columns (250×4.6 mm, 4 μm, Phenomenex, Torrance, Calif.) and singlewavelength or diode array UV detectors (set to 220 & 254 nm) connectedin series to a Bioscan FlowCount photomultiplier tube (PMT) (Bioscan,Wash., D.C.). Data was analyzed using the 32 Karat software package(Beckman-Colter). Mobile phase consisted of Solvent A: 0.05%trifluoroacetic acid in water and Solvent B: 100% acetonitrile, a flowrate of 1.5 mL/min, and a linear gradient beginning at 2 min afterinjection from 9% Solvent B then increasing to 81% over a 30 min periodunless otherwise stated.

During in-vitro experiments using alginate gel, molecular sieving highperformance liquid chromatography (HPLC) was performed on a WatersBreeze chromatography system with a Waters 2487 dual absorbance detector(220 & 320 nm) and a Bioscan Flow-count radioactivity detector. APhenomenex BioSep SEC-53000 column (7.8×300 mm) was eluted in isocratic0.1 M sodium phosphate, pH 6.8, at 1.0 mL/min.

The ⁶⁴Cu and ₁₁₁In-labeling yields were determined by radio-TLC, usingITLC-SG strips (Pall Life Sciences, Ann Arbor, Mich.) eluted with 200 mMEDTA in 0.9% aq. NaCl and performed using a Bioscan 200 imaging scanner(Bioscan, Wash. D.C.). In these conditions, free radionuclides migratewith Rf=0.9, while radionuclides attached to tetrazine 6 remain at theorigin.

PET/CT data was acquired using an Inveon Preclinical Imaging Station(Siemens Medical Solutions.

Animal Handling. All animals were handled in accordance with a protocolapproved by the University of California, Davis, Animal Use and CareCommittee.

Statistical analysis. Group variation is described as the mean± onestandard deviation. Single groups were compared with a two-tailedunpaired t test. Groups with P<0.05 were considered significantlydifferent. Microsoft Excel version 12.8.9 was used for all statisticalcalculations.

Example 1. Preparation of TCO-Modified Alginate 1

The preparation of TCO-modified alginate is described below, by firstpreparing the TCO-linker, and coupling to the alginate.

(R,E)-N-(2-aminoethyl)-2-(cyclooct-4-en-1-yloxy)acetamide (5). Compound3 (100 mg, 0.54 mmol), N-hydroxysuccinamide (69 mg, 0.60 mmol) andN,N′-dicyclohexylcarbodiimide (123 mg, 0.60 mmol) was dissolved inCH₂Cl₂ (2 mL) and stirred at rt for 2 h. The precipitated urea wasfiltered off through PVDF membrane. The membrane was washed with CH₂Cl₂(1 mL). The mother liquer was added to a sturring solution ofethylenediamine (324 mg, 5.4 mmol) in CH₂Cl₂ (10 mL). The reaction wasstirred at rt for 2 h. The reaction mixture was washed with water (2×10mL). The organic layer was dried with MgSO₄ and concentrated underreduced pressure. The product was purified on preparative silica TLCusing a 4:1 mixture of CH₂Cl₂ and MeOH as a solvent. The yield ofcompound 5 was 60 mg (49%). ¹H NMR (CDCl3) δ 7.08 (bs, 1H), 5.61-5.45(m, 2H), 3.92 (q, J=11.23 Hz, 2H), 3.64 (dd, J1=9.91 Hz, J2=4.73 Hz,1H), 3.36 (q, J=6.07 Hz, 2H), 2.87 (bs, 2H), 2.31-2.16 (m, 4H),2.08-2.04 (m, 1H), 1.89-1.49 (m, 5H). ¹³C NMR (CDCl³) δ 170.34, 135.48,131.37, 75.80, 68.31, 41.28, 40.05, 34.28, 32.53, 29.74, 27.85. HRMS:m/z [M+H]⁺ calcd. for C₁₂H₂₃N₂O₂ 227.1754, found 227.1791.

Each gram of UP MVG alginate was combined with 176 μmol of TCO-amineunder standard carbodiimide chemistry conditions, as previouslydescribed for arginine-glycine-aspartic acid (RGD) andglycine-histidine-lysine (GHK) incorporation. The alginate product wasthen purified by dialysis against deionized water containing decreasingsalt concentrations for 4 days, frozen and lyophilized for 5-10 daysuntil dry. A 2.5% alginate solution was obtained by adding DPBS, andalginate gels were fabricated by the addition of calcium. Covalentmodification of alginate was confirmed through 1H-NMR studies (see FIGS.20-22). The same protocol without the TCO addition was used for theconstruction of control gels. The in vitro and in vivo studies were donewith TCO-gel from the exact same batch and used on the same day tominimize any variations in loading amount or loading efficiency.

For in vitro experiments, 800 μl of 2.5% alginate solution was mixedwith 200 μl of supersaturated Ca(SO4)2 solution (0.21 g of Ca(SO4)2 perml of double-distilled water (ddH2O)). The solutions were mixed for 30 susing a three-way stopcock to achieve a final alginate concentration of2%. The mixture was allowed to gel between two glass plates in acustom-made plastic model and incubated for 20 min at room temperature.The entire volume had a uniform appearance consistent with gelation. Thediscs were picked up with a spatula and weighed individually. Typically,a premade disc weighed approximately 100 mg and had roughly thefollowing dimensions: 8 mm (diameter) and 2 mm (height).

For in vivo use, the 2.5% alginate gel solution and the super-saturatedcalcium sulfate solution were mixed rapidly in the same proportions asmentioned above and immediately injected to the animal in the desiredamount.

Example 2. Preparation of Tetrazine-Modified Diagnostic Agent

Tz radioprobe 2 was synthesized as previously described (R. Rossin, P.R. Verkerk, S. M. van den Bosch, R. C. Vulders, I. Verel, J. Lub, M. S.Robillard, Angew. Chem. Int. Ed. Engl. 2010, 49, 3375).

Example 3. Preparation of (E)-cyclooct-2-envl 2-aminoethvlcarbamate

The title compound was prepared according to the procedure described inVersteegen, R. M. et. al., Angew. Chem. Int. Ed. 2013, 52, 14112-14116.

Example 4. Preparation of Releasable TCO-Modified Diagnostic Agent

Dissolved fluorescein-NHS ester (134 mg, 0.283 mmol) and(Z)-cyclooct-2-enyl 2-aminoethylcarbamate (60.0 mg, 0.283 mmol) in DMF(5 mL). Added triethylamine (77 μL, 0.566 mmol) and stirred at rt for 18h. Evaporated the solvent under high vacuum and redissolved the reactionmixture in methanol. Purified by preparatory thin layer chromatographyusing 1:9 MeOH:CH₂Cl₂ mixture as mobile phase. Yield=90 mg (55.6%). ¹HNMR (CD₃OD, 400 MHz) δ 8.42 (s, 1H), 8.17 (d, J=8.2 Hz, 1H), 7.25 (d,J=8.2 Hz, 1H), 6.86 (s, 2H), 6.57-6.50 (m, 4H), 5.78 (t, J=12.3 Hz, 1H),5.47 (d, J=16.4 Hz, 1H), 5.2 (app s, 1H), 3.61-3.31 (m, 5H), 2.32 (bs,1H), 1.98-1.88 (m, 3H), 1.83-1.77 (m, 1H), 1.69-1.04 (m, 5H), 0.83-0.75(m, 1H). ¹³C NMR (CD₃OD, 100 MHz) δ 168.74, 161.63, 158.85, 156.60,154.17, 137.94, 135.62, 132.86, 132.70, 130.20, 125.81, 125.31, 113.89,111.03, 103.81, 75.43, 41.69, 41.21, 37.08, 36.67, 30.14, 25.27. HRMS(ESI-MS) m/z: calcd. for C₃₂H₃₀N₂O₈ [M+H⁺] 571.2080; found 571.2025.

Example 5. Preparation of TCO-Modified Diagnostic Agent

Dissolved 2-((E)-cyclooct-2-enyloxy)-N-(2-aminoethyl)acetamide (50.0 mg,0.221 mmol) and fluorescein-NHS ester (105 mg, 0.221 mmol) in DMF (5mL). Added triethylamine (60 μL, 0.442 mmol) and stirred at rt for 18 h.Evaporated the solvent under high vacuum and redissolved the reactionmixture in methanol. Purified by preparatory thin layer chromatographyusing 1:9 MeOH:CH₂Cl₂ mixture as mobile phase. Yield=51 mg (39.5%). ¹HNMR (CD₃OD, 400 MHz) δ 8.46 (s, 1H), 8.21 (d, J=8.2 Hz, 1H), 7.65 (bs,1H), 7.28 (d, J=8.2 Hz, 1H), 6.69 (d, J=2.7 Hz, 2H), 6.58-6.50 (m, 4H),5.46-5.44 (m, 2H), 3.91 (d, J=5.5 Hz, 2H), 3.65-3.59 (m, 5H), 2.67 (s,1H), 2.32-2.26 (m, 2H), 2.32-2.26 (m, 2H), 2.20-2.13 (m, 1H), 2.08 (d,J=4.2 Hz, 1H), 1.96-1.92 (m, 1H), 1.79-1.67 (m, 3H), 1.54-1.46 (m, 1),1.30-1.25 (m, 1H), 1.23-1.15 (m, 1H). ¹³C NMR (CD₃OD, 100 MHz) δ 173.60,170.67, 168.69, 154.21, 137.21, 137.66, 136.66, 136.75, 132.53, 130.30,125.89, 125.09, 113.84, 110.98, 103.79, 77.56, 69.38, 41.31, 40.75,40.07, 39.94, 35.50, 33.58, 30.82, 29.05. HRMS (ESI-MS) calcd. forC₃₃H₃₂N₂O₈ [M+H⁺] 585.2237; found 585.2183.

Example 6. Preparation of Releasable TCO-Modified Amoxicillin

Dissolved (E)-cyclooct-2-enyl 4-nitrophenyl carbonate (100 mg, 0.343mmol) and amoxicillin (82.0 mg, 0.224 mmol) in DMF (5 mL). Addedtriethylamine (87 μL, 0.634 mmol) and stirred at rt for 18 h. Evaporatedthe solvent under high vacuum and redissolved the reaction mixture inmethanol. Purified by preparatory thin layer chromatography using 1:9MeOH:CH₂Cl₂ mixture as mobile phase. Yield=36 mg (31%). ¹H NMR (CD₃OD,400 MHz) δ 8.49 (bs, 1H), 7.57 (dd, J₁=21.8 Hz, J₂=8.2 Hz, 1H), 7.24 (d,J=6.8 Hz, 2H), 6.67 (d, J=6.9 Hz, 2H), 5.81-5.76 (m, 1H), 5.51 (d,J=16.4 Hz, 1H), 5.29 (s, 1H), 5.17 (s, 1H), 4.92 (s, 1H), 4.33 (s, 1H),3.62 (s, 3H), 3.38 (s, 1H), 2.50 (s, 2H), 2.38 (bs, 1H), 1.98-1.88 (m,3H), 1.81-1.75 (m, 1H), 1.64-1.53 (m, 2H), 1.37 (s, 3H), 1.19-1.12 (m,4H), 0.81-0.78 (m, 2H). ¹³C NMR (CD₃OD, 100 MHz) δ 170.49, 156.77,154.78, 132.16, 131.87, 131.24, 130.95, 128.60, 114.83, 72.99, 65.73,59.77, 59.07, 57.53, 57.03, 56.89, 51.93, 48.60, 35.54, 35.31, 28.46,27.07, 26.76, 23.67, 23.54. HRMS (ESI-MS) m/z: calcd. for C₃₃H₃₂N₂O₈[M+MeO⁻] 548.2072; found 548.2042.

Example 7. Preparation of TCO-Modified Amoxicillin

Dissolved 2-((E)-cyclooct-2-enyloxy)acetic acid (85.0 mg, 0.461 mmol),N-hydroxysuccinamide (53.0 mg, 0.461 mmol) andN,N′-dicyclohexylcarbodiimide (95.0 mg, 0.461 mmol) in CH₂Cl₂ (5 mL).Stirred at rt for 18 h. The precipitate was filtered and the supernatantwas concentrated under reduced pressure. Added a solution of amoxicillin(160 mg, 0.438 mmol) in DMF (10 mL) and stirred at rt for 18 h.Evaporated the solvent under high vacuum and redissolved the reactionmixture in methanol. Purified by preparatory thin layer chromatographyusing a 1:9 MeOH:CH₂Cl₂ mixture as mobile phase. Yield=20 mg (8.6%). ¹HNMR (CD₃OD, 400 MHz) δ 7.33 (t, J=9.5 Hz, 1H), 7.24 (t, J=0.6 Hz, 1H),6.76 (d, J=8.2 Hz, 2H), 5.68-5.45 (m, 4H), 4.32 (bs, 1H), 3.99-3.09 (m,2H), 3.72 (s, 1H), 3.47 (bs, 1H), 2.67 (s, 6H), 2.37-1.96 (m, 5H),1.85-1.80 (m, 3H), 1.56 (s, 3H), 1.47 (s, 3H), 1.28-1.17 (m, 3H). ¹³CNMR (CD₃OD, 100 MHz) δ 175.08, 172.65, 171.82, 158.81, 131.16, 131.07,130.69, 130.13, 129.98, 129.89, 116.69, 83.19, 68.67, 66.71, 59.88,58.86, 57.05, 53.13, 35.26, 35.14, 34.26, 34.19, 27.18, 26.75, 26.69,26.41, 23.44, 23.41. HRMS (ESI-MS) m/z: calcd. for C₃₃H₃₂N₂O₈ [M+MeO⁻]548.2072; found 548.2042.

Example 8. Preparation of Modified Agarose

The NHS-activated agarose spin columns were purchased from Pierce/ThermoFisher Scientific (Rockford, Ill.). The amine precursors to 1a or 1b ofFIG. 16B were coupled to the agarose beads using the manufacturer'srecommended protocol. Briefly, 33 mg of dry agarose was incubated with 4μmol of the amine precursor of 1a or 1b in PBS buffer pH 7.4 for 3 hwith gentle mixing. The agarose beads were washed several times withPBS. The amount of 1a or 1b bound to the column was estimated bymonitoring the absorbance at 520 nm of the supernatants obtained fromthe washes. The unreacted NHS groups on agarose were capped with 1 MTris, pH 7.4.

Example 9. Kinetics of Catch & Release Linker

The agarose beads modified with either 1a or 1b, using the methoddescribed above, were treated with 2 μmol of TCO-R-Rhodamine((E)-5-((2-(((cyclooct-2-en-1-yloxy)carbonyl)amino)ethyl)carbamoyl)-2-(3-(dimethyl-□⁴-azanylidene)-6-(dimethylamino)-3H-xanthen-9-yl)benzoicacid) for 2 min. The supernatant was collected after a quickcentrifugation and the agarose was resuspended in water. Thesupernatents were collected at regular time intervals and analyzed byESI-MS.

The samples were analyzed on a Thermo Fisher Scientific (West PalmBeach, Calif.) LTQ Orbitrap Velos Mass spectrometer, using quartzcapillary emitters. To facilitate spray optimization, 10% isopropylalcohol was added to each sample prior to MS analysis. The releaseproduct, Rhodamine ethyl diamine(5-((2-aminoethyl)carbamoyl)-2-(3-(dimethyl-□⁴-azanylidene)-6-(dimethylamino)-3H-xanthen-9-yObenzoicacid), was analyzed in the positive mode. The release product rhodamineethyl diamine was independently synthesized for ESI-MS calibration(Figure SX). The calibration curve was used to estimate the amount ofrhodamine ethyl diamine in the supernatants of each step.

Example 10. Preparation of Modified Tz-Me-Alginate

Each gram of UP MVG alginate was combined with 176 μmoles of(4-(6-Methyl-1,2,4,5-tetrazin-3-yOphenyOmethanamine (Tz-Me-amine) understandard carbodiimide chemistry conditions as previously described forRGD, GHK and TCO amine incorporation. Then the alginate product waspurified by dialysis against deionized water containing decreasing saltconcentrations for 4 days, frozen and lyophilized for 5-10 days untildry. A 2.5% alginate solution was obtained by adding ddH2O, and alginategels were fabricated by the addition of calcium. Covalent modificationof alginate was confirmed through 1H-NMR studies (see FIG. 23). The sameprotocol without the TCO addition was used for the construction ofcontrol gels. The in-vitro and in-vivo studies were done with TCO-Gel 1from the exact same batch and used on the same day to minimize anyvariations in loading amount or loading efficiency.

For in-vivo experiments, 800 μl of 2.5% alginate solution were mixedwith 200 μl of supersaturated Ca(SO4)₂ solution (0.21 g Ca(SO4)₂/mlddH2O). The solutions were mixed for 30 s using a three-way stopcock toachieve a final alginate concentration of 2%. The mixture wasimmediately injected to the animal in the desired amount.

Example 11. In-Vivo Evaluation of Catch & Release Linker

After IACUC approval, in-vivo real-time biodistribution studies offluorescence were carried out in nu/nu mice (n=2 per condition) byinjecting either nothing or a type of alginate (control vs Tz-Gel). Thensubjects received a tail-vein injection of TCO-R-F or TCO-NR-F. Thenegative controls were: 1. No gels with TCO-R-F (Negative control, mouse1); 2. control alginate with TCO-R-F (Gel control, mouse 4). The twoexperimental groups were Tz-gel and either TCO-R-F (Released protocol,mouse 2) or TCO-NR-F (Immobilized protocol, mouse 3). Fluorescence wasmeasured with an IVIS Spectrum (Perkin Elmer, Mass.) and reported inradiance.

Example 12. Minimum Inhibitory Concentration (MIC) of ReleasableVancomycin

We created serial dilutions of vancomycin or TCO-R-Vanco with either aregular alginate gel or Tz-gel overnight in ddH₂O. The following dayluminescent methicillin sensitive Staph. aureus (MSSA, Xen 29, PerkinElmer, Mass.) in 2.2% Mueller Winto broth were added to the mixture. Theplates were then placed in the incubator for 24 and allowed to grow for24 hours (n=3). Then luminescence was measured with an IVIS Spectrum(Perkin Elmer, Mass.) and reported in radiance.

Example 13. Preparation of Releasable TCO-Rhodamine

The Rhodamine-NHS ester was synthesized as described by Brunet, A.;Aslam, T.; Bradley, M. Bioorg. Med. Chem. Lett. 2014, 24, 3186-3188.Dissolved rhodamine-NHS ester (50 mg, 0.095 mmol) and(E)-cyclooct-2-enyl-2-aminoethylcarbamate (40.0 mg, 0.190 mmol) inCH2Cl2 (5 mL). Added triethylamine (129 μL, 0.95 mmol) and stirred at rtfor 18 h. Evaporated the solvent under high vacuum and redissolved thereaction mixture in methanol. Purified by preparatory thin layerchromatography using 7.5:2.5:90 MeOH:Et3N:CH2Cl2 mixture as mobilephase. Yield=28 mg (47%). ¹H NMR (CD₃OD, 400 MHz) δ 8.54 (s, 1H), 8.04(d, J=8.2 Hz, 1H), 7.34 (d, J=8.2 Hz, 1H), 7.23 (d, J=9.6 Hz, 1H), 6.99(dd, J1=2.7 Hz, J2=9.5 Hz, 1H), 6.89 (d, J=2.8 Hz, 1H), 5.85 (t, J=13.7Hz, 1H), 5.55 (d, J=16.4 Hz, 1H), 5.26 (s, 1H), 3.67-3.36 (m, 4H),3.32-3.21 (m, 9H), 2.93-2.77 (m, 6H), 2.49-2.36 (m, 1H), 2.10-1.79 (m,5H), 1.78-1.41 (m, 4H), 1.39-1.25 (m, 1H), 1.22-1.06 (m, 10H), 0.93-0.79(m, 1H). ¹³C NMR (CD3OD, 100 MHz) δ 172.47, 169.41, 161.68, 159.08,158.76, 142.06, 137.19, 132.96, 132.70, 130.90, 129.85, 129.63, 115.09,114.86, 97.53, 76.85, 75.38, 41.96, 41.78, 41.01, 37.18, 36.92, 30.21,25.35. HRMS (ESI) m/z: calcd. for C36H41N4O6 [M+1]+ 625.3026; found625.2976.

Example 14. Preparation of Non-Releasable TCO-Rhodamine

Dissolved rhodamine-NHS ester (50 mg, 0.095 mmol) and2-((E)-cyclooct-4-enyloxy)-N-(2-aminoethyl)acetamide (43.0 mg, 0.190mmol) in CH2Cl2 (5 mL). Added triethylamine (129 μL, 0.95 mmol) andstirred at rt for 18 h. Evaporated the solvent under high vacuum andredissolved the reaction mixture in methanol. Purified by preparatorythin layer chromatography using 7.5:5:2.5:90 MeOH:Et₃N:CH₂Cl₂ mixture asmobile phase. Yield=35 mg (58%). 1H NMR (CD₃OD, 400 MHz) δ 8.57 (s, 1H),8.07 (d, J=8.2 Hz, 1H), 7.34 (d, J=8.2 Hz, 1H), 7.22 (d, J=8.2 Hz, 2H),6.97 (dd, J1=2.7 Hz, J2=9.6 Hz, 2H), 6.88 (d, J=2.7 Hz, 2H), 5.61-5.43(m, 2H), 3.94 (d, J=4.1 Hz, 2H), 3.69-3.55 (m, 6H), 3.31 (s, 2H),2.41-2.31 (m, 2H), 2.27-2.14 (m, 2H), 2.03-1.95 (m, 1H), 1.90 (s, 2H),1.84-1.71 (m, 4H), 1.61-1.50 (m, 2H). 13C NMR (CD₃OD, 100 MHz) δ 173.49,172.35, 169.46, 161.47, 159.04, 158.71, 142.12, 136.97, 136.88, 132.70,132.57, 129.84, 129.65, 115.02, 114.83, 97.55, 77.65, 69.44, 41.40,41.00, 35.58, 33.62, 30.88, 29.10. HRMS (ESI) m/z: calcd. for C37H43N4O6[M+1]+ 639.3183; found 639.3151.

Example 15. Rhodamine Release Product

Dissolved rhodamine-NHS ester (20.0 mg, 0.0379 mmol) andN-Boc-ethylenediamine (13.2 mg, 0.0758 mmol) in CH2Cl2 (5 mL). Addedtriethylamine (51 (L, 0.379 mmol) and stirred at rt for 18 h. Evaporatedthe solvent under high vacuum and redissolved the reaction mixture inmethanol. Purified by preparatory thin layer chromatography using10:5:85 MeOH:Et₃N:CH₂Cl₂ mixture as mobile phase. The isolated productwas treated with a 4:1 mixture of CH₂C₂: TFA (5 mL) for 1 h. Yield=18 mg(81%). ¹H NMR (CD3OD, 400 MHz)™ 8.83 (s, 1H), 8.32 (d, J=9.6 Hz, 1H),7.55 (d, J=8.1 Hz, 1H), 7.13 (d, J=9.6 Hz, 2H), 7.05 (dd, J1=6.8 Hz,J2=2.7 Hz, 2H), 6.96 (d, J=2.7 Hz, 2H), 3.77 (t, J=5.4 Hz, 2H), 3.30 (s,12H), 3.22-3.17 (m, 2H). ¹³C NMR (CD3OD, 100 MHz)™ 169.28, 167.47,160.69, 159.10, 138.52, 132.63, 132.11, 132.02, 131.67, 115.71, 114.85,97.63, 41.08, 40.97, 39.06. HRMS (ESI) calcd. for C₂₇H₂₉N₄O₄ [M+1]+473.2189; found 473.2141.

Example 16. Releasable TCO-Vancomycin

Dissolved vancomycin (50.0 mg, 0.0345 mmol) and triethylamine (20 μL,0.145 mmol) in water (1 mL). Added a solution of (E)-cyclooct-2-enyl4-nitrophenyl carbonate (19.0 mg, 0.0652 mmol) in DMF (25 μL). Stirredthe reaction mixture at 40° C. for 18 h. Filtered out the precipitatethrough a 45 μm PTFE membrane. The supernatant was subjected to HPLCpurification to obtain the title compound. A gradient of 10-60% CH3CN inH2O was used for the HPLC purification. Yield=3 mg (5.5%). 1H NMR(CD3OD, 400 MHz)σ7.61 (s, 1H), 7.55-7.38 (m, 2H), 7.19 (bs, 1H), 7.03(s, 1H), 6.81 (bs, 1H), 6.44 (s, 1H), 6.38 (s, 1H), 6.05 (bs, 1H),5.55-5.37 (m, 2H), 5.32 (s, 1H), 5.23 (s, 1H), 4.68 (s, 36H), 4.52-4.31(m, 2H), 4.15 (s, 1H), 4.00 (t, J=8.2 Hz, 1H), 3.85-3.61 (m, 3H), 3.56(t, J=8.2 Hz, 1H), 3.35 (s, 1H), 2.67 (s, 4H), 2.03-1.88 (m, 2H),1.75-1.39 (m, 3H), 1.33 (s, 3H), 1.05 (s, 3H), 0.75 (dd, J1=8.2 Hz,J2=13.7 Hz, 6H). HRMS (ESI) m/z: calcd. for C75H87Cl2N9O26 [M+1]+1602.4660; found 1602.5256.

Example 17. Releasable TCO-Daptomycin

Daptomycin (100 mg, 0.062 mmol) was dissolved in water (1 mL). Asolution of (E)-cyclooct-2-enyl 4-nitrophenyl carbonate (36 mg, 0.123mmol) in DMF (50 μL) was added, followed by Triethylamine (167 mg, 1.65mmol). The reaction mixture was stirred at rt for 18 h. The titleproduct was obtained as a white foam after HPLC purification using asemipreparative Phenomenex Luna 5u C18 (2) column and a gradient of10-65% CH₃CN in H₂O. Yield=44 mg (40%). ¹H NMR (CD₃OD, 400 MHz) δ 7.66(bs, 2H), 7.60-7.46 (m, 2H), 7.39 (s, 1H), 7.25-7.11 (m, 2H), 7.02 (s,1H), 6.96 (s, 1H), 6.83-6.78 (m, 1H), 6.56 (s, 1H), 6.00-5.68 (m, 4H),5.68-5.57 (m, 1H), 5.54 (s, 1H), 5.51-5.26 (m, 8H), 5.14 (bs, 1H), 4.96(bs, 1H), 4.67 (s, 1H), 4.64 (s, 1H), 4.58 (s, 1H), 4.14 (s, 1H),3.88-3.79 (m, 2H), 3.78-3.71 (m, 1H), 3.59-3.43 (m, 2H), 3.19 (q, J=8.2Hz, 7H), 2.95 (bs, 4H), 2.59-2.32 (m, 5H), 2.23-1.76 (m, 15H), 1.56-1.44(m, 8H), 1.30 (t, J=8.1 Hz, 10H), 1.23-1.16 (m, 5H), 1.00-0.87 (m, 9H).MS (ESI) m/z: calcd. for C₇₅H₈₇Cl₂N₉O₂₆ [M+H+MeCN]⁺ 1813.82; found1813.80.

Example 18. Releasable TCO-Doxorubicin

(E)-cyclooctene doxorubicin conjugate was synthesized as described byVersteegen, R. M. et. al., Angew. Chem. Int. Ed. 2013, 52, 14112-14116.The spectra from ¹H NMR (CDCl₃) matched the published data.

Example 19. Releasable TCO-Cyclic AMP

Cyclic AMP (80 mg, 0.243 mmol) and (E)-cyclooct-2-enyl 4-nitrophenylcarbonate (142 mg, 0.486 mmol) were dissolved in anhydrous DMF (8 mL).4-Dimethylaminopyridine (238 mg, 1.94 mmol) was added and the reactionmixture was stirred at 30° C. for 18 h. The solvent was removed underhigh vacuum and the title product was obtained as a white foam afterpreparative column chromatography using 15% MeOH in CH₂Cl₂ as a mobilephase. Yield=45 mg (38%). ¹H NMR (CDCl₃, 400 MHz) δ 7.61 (s, 1H),7.57-7.41 (m, 2H), 7.19 (bs, 1H), 7.03 (s, 1H), 6.81 (bs, 1H), 6.44 (s,1H), 6.38 (s, 1H), 6.06 (bs, 1H), 5.53-5.39 (m, 2H), 5.32 (s, 2H), 5.24(s, 1H), 4.48 (bs, 2H), 4.16 (s, 1H), 4.00 (t, J=6.8 Hz, 1H), 3.83-3.61(m, 3H), 3.60-3.50 (m, 1H), 3.35 (s, 1H), 2.68 (s, 4H), 2.03-1.90 (m,2H), 1.75-1.40 (m, 3H), 1.33 (s, 3H), 1.05 (s, 3H), 0.74 (dd, J1=8.2 Hz,J2=13.6 Hz, 6H). HRMS (ESI) m/z: calcd. for C₁₉H₂₅N₅O₈P [M+1]⁺ 482.1441;found 482.1461.

Example 20. Releasable TCO-Compound 3

Compound 3 (132 mg, 0.413 mmol) and (E)-cyclooct-2-enyl 4-nitrophenylcarbonate (80 mg, 0.275 mmol) were dissolved in anhydrous DMF (5 mL).Triethylamine (167 mg, 1.65 mmol) was added and the reaction mixture wasstirred at rt for 18 h. The solvent was removed under high vacuum andthe title product was obtained as a white foam after preparative columnchromatography using 10% MeOH in CH₂Cl₂ as a mobile phase. Yield=70 mg(36%). ¹H NMR (CD₃OD, 400 MHz) δ 8.28 (s, 1H), 5.93 (d, J=6.9 Hz, 1H),5.89-5.78 (m, 1H), 5.54 (bs, 1H), 5.28 (bs, 1H), 4.73 (t, J=5.5 Hz, 1H),4.37-4.31 (m, 1H), 4.23-4.13 (m, 3H), 3.92 (d, J=10.9 Hz, 1H), 3.73 (d,J=12.3 Hz, 1H), 3.65-3.40 (m, 1H), 3.35 (s, 1H), 3.31 (s, 1H), 2.47-2.30(m, 1H), 2.10-1.75 (m, 4H), 1.75-1.57 (m, 2H), 1.53 (m, 1H), 1.26 (bs,1H), 1.18-1.06 (m, 2H), 0.91-0.74 (m, 1H). HRMS (ESI) m/z: calcd. forC₂₂H₂₈N₆O₆ [M+1]⁺ 473.2149; found 473.2117.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, one of skill in the art will appreciate that certainchanges and modifications may be practiced within the scope of theappended claims. In addition, each reference provided herein isincorporated by reference in its entirety to the same extent as if eachreference was individually incorporated by reference. Where a conflictexists between the instant application and a reference provided herein,the instant application shall dominate.

What is claimed is:
 1. A bioactive composition comprising: a therapeuticagent other than doxorubicin, vancomycin, and paclitaxel; a bindingagent comprising a trans-cyclooctene; and a linker comprising from about1 to about 10 linking atoms, covalently linking the binding agent to thetherapeutic agent.
 2. The composition of claim 1, wherein the linkercomprises the structure of:


3. The composition of claim 1, wherein the binding agent and the linkertogether have the structure selected from the group consisting of


4. The bioactive composition of claim 1, wherein the therapeutic agentis daptomycin, irinotecan, SN-38, amoxicillin, cyclic AMP, doxycyclin,amphotericin, ixabepilone, podophyllotoxin, patupilone, or rapamycin. 5.The bioactive composition of claim 4, wherein the therapeutic agent isdaptomycin.
 6. The bioactive composition of claim 4, wherein thetherapeutic agent is irinotecan.
 7. The bioactive composition of claim4, wherein the therapeutic agent is SN-38.
 8. The bioactive compositionof claim 4, wherein the therapeutic agent is amoxicillin.
 9. Thebioactive composition of claim 4, wherein the therapeutic agent iscyclic AMP.
 10. The bioactive composition of claim 4, wherein thetherapeutic agent is doxycyclin.
 11. The bioactive composition of claim4, wherein the therapeutic agent is amphotericin.
 12. A bioactivecomposition comprising a therapeutic agent other than doxorubicin,wherein the therapeutic agent is linked by a covalent bond to astructure


13. The bioactive composition of claim 12, wherein the therapeutic agentis vancomycin, paclitaxel, daptomycin, etoposide, irinotecan, SN-38,amoxicillin, cyclic AMP, doxycyclin, amphotericin, ixabepilone,podophyllotoxin, patupilone, or rapamycin.
 14. The bioactive compositionof claim 13, wherein the therapeutic agent is vancomycin.
 15. Thebioactive composition of claim 13, wherein the therapeutic agent ispaclitaxel.
 16. The bioactive composition of claim 13, wherein thetherapeutic agent is daptomycin.
 17. The bioactive composition of claim13, wherein the therapeutic agent is SN-38.
 18. A bioactive compositioncomprising: a diagnostic agent; a binding agent comprising atrans-cyclooctene; and a linker comprising from about 1 to about 10linking atoms, covalently linking the binding agent to the diagnosticagent.
 19. The bioactive composition of claim 18, wherein the diagnosticagent is an optical probe.